L-amino acids are used in animal nutrition, in the food industry, in human medicine and in the pharmaceuticals industry. It is known that these amino acids are produced by fermentation of strains of coryneform bacteria in particular Corynebacterium glutamicum. Due to their great significance, efforts are constantly being made to improve the production process. Improvements to the process may relate to measures concerning fermentation technology, for example stirring and oxygen supply, or to the composition of the nutrient media, such as for example sugar concentration during fermentation, or to working up of the product by, for example, ion exchange chromatography, or to the intrinsic performance characteristics of the microorganism itself.
The performance characteristics of these microorganisms are improved using methods of mutagenesis, selection and mutant selection. In this manner, strains are obtained which are resistant to antimetabolites, such as for example the lysine analogue S-(2-aminoethyl)cysteine, or are auxotrophic for regulatorily significant amino acids and produce L-amino acids. For some years, the methods of recombinant DNA technology have also been used for strain improvement of strains of Corynebacterium which produce L-amino acid.
LRP (leucine-responsive protein) is a global regulator, first described in Escherichia coli, which influences the transcription of a series of genes, the gene products of which are involved in the transport, biosynthesis and degradation of amino acids (Calvo et al, Microbiol. Rev. 58:466–490 (1994)). In recent years, similar genes have also been identified in other organisms, such as Bradyrhizobium japonicum (King, et al., J. Bacteriol. 179:1828–1831 (1997)), Klebsiella aerogenes (Janes, et al, J. Bacteriol. 181:1054–1058 (1999)), Sulfolobus acidocaldarius (Charlier et al., Gene 201:63–68 (1997)) and in the gram positive bacterium Bacillus subtilis (Belitsky, et al., J. Bacteriol. 179:5448–5457 (1997)).
In E. coli, the lrp protein regulates its own expression. Lrp also has an either negative or positive influence on many genes in E. coli. In general, it is the expression of gene products which are active in biosynthetic pathways which is stimulated. Gene products having a catabolic action are generally correspondingly negatively controlled. In some cases, the action of lrp is potentiated by the addition of L-leucine, but addition of L-leucine may also have a negative effect (Newman, et al., In: Neidhardt et al. Escherichia coli and Salmonella typhimurium: Cellular and molecular biology, American Society for Microbiology, Washington D.C., pp. 1513–1525 (1996)). In E. coli, lrp regulates a large number of genes and operons which play a central role in amino acid biosynthesis and amino acid catabolism. The following operons in E. coli are, for example, negatively controlled: livJ, which codes for a binding protein in a highly sophisticated uptake system for branched-chain amino acids (Haney, et al., J. Bacteriol. 174:108–115 (1992)) and lysU, which codes for lysine tRNA synthetase (Gazeau, et al., FEBS Letters 300:254–258 (1994)). Genes which have hitherto been known to be positively influenced in E. coli include, inter alia, ilvIH, gltBDF and leuABCD (Lin, et al., J. Bacteriol. 174:1948–1955 (1992)).
The last-stated operon is of fundamental interest in leucine biosynthesis and of particular interest for lysine biosynthesis in Corynebacterium glutamicum. It is suspected that there is an association between leucine auxotrophy and elevated lysine productivity values (Schrumpf et al. Appl. Microbiol. Biotech. 37:566–571 (1992)). Patek, et al. (Appl. Environ. Microbiol. 60:133–140 (1994)) have demonstrated that inactivating leuA in some lysine producers of C. glutamicum results in increased lysine yields. In a mutant of Brevibacterium lactofermentum, Tosaka, et al. (Agri. Biol. Chem. 43:265–270 (1979)) have been able to achieve a reduction in lysine formation with a simultaneous increase in threonine formation by addition of L-leucine.